Chimaeric vector system

ABSTRACT

This invention relates to a process for producing a Simian Immunodeficiency Virus (SIV) encoding a heterologous gene, which process comprises infecting a host cell with a first vector which is capable of producing SIV capsid and a second vector comprising a Human Immunodeficiency Virus type 2 (HIV-2) packaging signal sufficient to package the second vector in the SIV capsid and a heterologous gene capable of being expressed by the vector; and culturing the host cell.

FIELD OF THE INVENTION

This invention relates to vectors and their use in gene transfer. Thevectors are based on retroviruses, adapted so that they cannot packagetheir own RNA, and which can be used as infectious agents to transferforeign genes, e.g. for somatic gene therapy.

BACKGROUND OF THE INVENTION

Modified viruses have been used to deliver genetic material to cells,both for research/development purposes and for clinical purposes. Someof the most successful gene transfer systems (‘vectors’) are based onretroviruses, and more recently, on lentiviruses, a subfamily ofretrovirideae. Retroviral vectors have the advantages of being able toefficiently infect a broad range of cell types, and of being able tointegrate the genetic material they carry (e.g. exogenous therapeuticgenes) into the genome of the target cell (e.g. cells of the humanpatient). However, retroviral vectors can only infect dividing cells,and this limits their use.

Lentiviral vectors have a number of advantages over retroviral vectorsincluding the ability to infect both dividing and non-dividing cells.

However, for both retroviral and lentiviral vectors there are concernsthat the genetic homology between the packaging constructs and theconstructs comprising the packageable vectors and/or other viralsequences, including sequences present in the cells in which theretroviral vectors are produced, could lead to recombination events thatcould generate a dangerous replicating virus.

These recombination events are particularly prone to occur in the cellline in which the vector is produced. This is because, in order for thecell line to produce the vector, it must contain certain viral sequenceswhich express the proteins and other factors necessary to package thevector into a virus-like particle that then can infect cells, reversetranscribe RNA and integrate the proviral DNA into the host cell genome.Recombination between the vector and these ‘helper’ sequences may intheory produce a dangerous replicating virus.

Testing of lentiviral vector biosafety in appropriate animal models is amajor concern associated with the use of lentiviral vectors in clinicaltrials. As HIV-1 only causes AIDS in humans, there is presently noanimal model to test the safety of HIV-1 based vectors.

SUMMARY OF THE INVENTION

The present inventors have surprising found that a non-reciprocityexists between HIV-2 and SIV such that SIV Gag proteins can captureHIV-2 RNA vectors but that the reverse cannot occur. Using apackaging-defective SIV provirus vector, packaging-defective cell linesmay be produced which generate chimaeric SIV/HIV-2 vectors for efficientintroduction of a desired gene or genetic sequence into mammalian cells.

One aspect of the invention provides a process or method of producing avirus, in particular a chimaeric virus for use in gene therapy,comprising;

-   -   culturing a host cell which comprises one or more Simian        Immunodeficiency Virus (SIV) nucleic acid sequences capable of        producing an SIV capsid and which further comprises a vector        comprising a Human Immunodeficiency Virus type 2 (HIV-2)        packaging signal and a heterologous nucleic acid sequence;    -   said vector being packaged in the SIV capsid to produce a viral        particle comprising the heterologous nucleic acid sequence.

In some embodiments, a method may comprise infecting the host cell whichproduces the SIV capsid with the vector.

In other embodiments, a host cell may be infected with a first vectorwhich comprises the one or more Simian Immunodeficiency Virus (SIV)nucleic acid sequences capable of producing an SIV capsid and a secondvector which comprises the human Immunodeficiency Virus type 2 (HIV-2)packaging signal and a heterologous nucleic acid sequence.

Accordingly, another aspect of the invention provides a process forproducing a Simian Immunodeficiency Virus (SIV) encoding a heterologousgene, which process comprises infecting a host cell with a first vectorwhich is capable of producing SIV capsid and a second vector comprisinga Human Immunodeficiency Virus type 2 (HIV-2) packaging signalsufficient to package the vector in the SIV capsid and a heterologousgene capable of being expressed by the vector; and culturing the hostcell.

Another aspect of the invention provides a process for making a producercell for the generation of virus comprising:

-   -   infecting a host cell which comprises one or more Simian        Immunodeficiency Virus (SIV) nucleic acid sequences capable of        producing an SIV capsid with a vector comprising a Human        Immunodeficiency Virus type 2 (HIV-2) packaging signal and a        heterologous nucleic acid sequence.

The invention also extends to host cells and viruses produced by theprocesses of the invention and kits and vector systems for use in suchmethods. Pharmaceutical compositions may be formulated which comprisesuch host cells or viruses.

The viruses, nucleic acids and cells of the invention may be used ingene therapy. Thus, the invention provides a method of delivering atherapeutic or antigenic protein or peptide to an individual comprisingadministering to the individual an effective amount of a first andsecond vector as described above, a virus, nucleic acid or cellaccording to the invention, or a pharmaceutical composition according tothe invention.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the cross packaging efficiency of HIV-1 gag-pol (see table2).

FIG. 2 shows the cross packaging efficiency of SIV gag-pol (see table3).

FIG. 3 shows the cross packaging efficiency of HIV-2 gag-pol (see table4).

FIG. 4 shows the SIVmac leader region.

FIG. 5 shows a comparison of HIV-1, HIV-2 and SIV leader sequenceregions with localization of the major packaging signal. Numbering isfrom RNA cap size and not the 5′ LTR.

DESCRIPTION OF THE INVENTION

Packaging-defective proviral constructs are systems in which theprovirus is capable of producing some viral proteins but is notreplication-competent because the viral RNA cannot be packaged intovirions. These constructs are commonly used to create packaging celllines. The packaging defective proviral construct or constructs areknown as the ‘packaging constructs’. The RNA transcripts of thepackaging constructs do not contain the sequences required forrecognition and encapsidation into a viral particle. Introduction orexpression of heterologous RNA transcripts containing the necessarypackaging signal sequences into packaging cell lines results in theheterologous RNA being packaged into virions. A packaging cell linewhich produces virions comprising heterologous RNA is known as aproducer line.

A producer line may, for example, contain the gag-pol sequences from SIVand a vector (i.e. a sequence of nucleic acid containing a packagingsignal) derived from HIV-2. The producer line may also contain asequence encoding an envelope protein from a non-SIV source. Suitableenvelope proteins may be obtained from a variety of sources including,but not limited to, ecotropic retroviruses, amphotropic retroviruses,vesicular stomatitis virus (VSV) or any other kind of virus.

The env sequences may be part of the SIV proviral construct or, morepreferably, may be located on a separate plasmid construct under controlof a separate promoter and polyadenylation sequence in order to reducehomology and the possibility of recombination events.

The producer line may also contain one or more sequences encoding otherproteins including but not limited to antibodies and antibody-likemolecules, and any epitopes or sub-units thereof, and any modifiedversions or fragments of any of the above. Modifications may include,but are not limited to, general post-translational protein modificationssuch as glycosylation (which might be dependent on the expression ofnative or exogenous glycosyltransferases or glycosylases or otherenzymes in the producer line or in the supernatant).

The affinity of this gene transfer system for target cells may beprovided by the envelope sequences, while the efficient reversetranscription and integration functions may be provided by the SIVgag-pol. Efficient packaging may be provided by a combination of the SIVgag-pol and the HIV-2 vector sequences. This combination would give riseto superior gene transfer efficiency as compared with many othersystems.

The use of gag-pol and vector sequences from SIV and HIV-2 (and,optionally, an envelope gene from a third source) respectively may beuseful in reducing the recombination probability, thereby increasing thesafety of the system relative to other viral vector systems.

The superior safety and efficiency features derived from the combinationof SIV and HIV-2 sequences, combined with the general advantages oflentiviral vectors (e.g. stable, long-term expression in dividing andnon-dividing human cells and minimal disruption of the endogenous humangenetic material) provide a gene transfer system with improved safety,efficiency and stability of expression in human cells.

Packaging Defective SIV Vectors

SIV packaging-defective vectors may be produced using standardtechniques. The region between the primer-binding site and the 5′ majorsplice donor in SIV contains sequences necessary for efficient packagingof SIV RNA into virions (Strappe, P.M. et al. J. Gen Virol (2003)84:2423-2430). In addition, the region between the 5′ major splice donorand the gag initiation codon contains a second and less importantregion, important but not essential for packaging of SIV RNA intovirions. A vector comprising a packaging-defective SIV provirus may beprepared wherein the vector contains a nucleotide sequence whichcorresponds to a sufficient number of nucleotides from an SIV genome toexpress desired SIV products, but does not correspond to a sufficientnumber of nucleotides corresponding to the region between theprimer-binding site and the 5′ major splice donor or between the splicedonor and the gag initiation codon to efficiently package SIV RNA (thepackaging sequence). In other words, a vector may comprise SIV nucleicacid sequences which allow expression of desired SIV products, inparticular SIV gag-pol, but may not comprise SIV nucleic acid sequenceswhich allow efficient packaging of SIV RNA.

These sequences preferably correspond to the genome of SIV. The term‘corresponds’ means that conservative additions, deletions andsubstitutions are permitted. The primer-binding site (23 bp) and the 5′major splice donor are respectively numbered 121-143 and 295-296 in theSIV genomic nucleotide sequence, where the transcript start site isdefined as 1. In the SIVmac32H sequence (Acc No D01065) theprimer-binding site and the 5′ major splice donor are respectivelynumbered 822-849 and 985-986, where the first nucleotide of the 5′LTR isdefined as 1. The genomic sequences of examples of other strains of SIVare set out in Table 5.

In particular, an SIV genome as used herein refers to the viral RNAderived from an SIV. The simian immunodeficiency viruses.(SIV) of theinvention may be derived from any SIV strain, for example a strainhaving a genomic sequence set in Table 5, or derivatives thereof.Derivatives preferably have at least 70% sequence homology to the SIVgenome, more preferably at least 80%, even more preferably at least 90or 95%.

Sequence homology may also be expressed in terms of sequence similarityor sequence identity. Sequence similarity and identity are commonlydefined with reference to the algorithm GAP (Genetics Computer Group,Madison, Wis.). GAP uses the Needleman and Wunsch algorithm to align twocomplete sequences that maximizes the number of matches and minimizesthe number of gaps. Generally, default parameters are used, with a gapcreation penalty=12 and gap extension penalty=4. Use of GAP may bepreferred but other algorithms may be used, e.g. BLAST (which uses themethod of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA(which uses the method of Pearson and Lipman (1988) PNAS USA 85:2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981)J. Mol Biol. 147: 195-197), or the TBLASTN program, of Altschul et al.(1990) supra, generally employing default parameters. In particular, thepsi-Blast algorithm (Nucl. Acids Res. (1997) 25 3389-3402) may be used.Sequence identity and similarity may also be determined usingGenomequest™ software (Gene-IT, Worcester Mass. USA).

Similarity allows for “conservative variation”, i.e. substitution of onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another, or the substitution of one polar residue for another, suchas arginine for lysine, glutamic for aspartic acid, or glutamine forasparagine. Particular amino acid sequence variants may differ from aknown polypeptide sequence as described herein by insertion, addition,substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20 20-30,30-50, or more than 50 amino acids.

Sequence comparisons are preferably made over the full-length of therelevant sequence described herein.

Other derivatives which may be used to obtain the viruses of the presentinvention include strains that already have mutations in some SIV genessuch as a mutation in the nef gene as described in Rud et al 1994 J. GenVirol 75, 529-543. Other mutations may also be present as set out inmore detail below. The position of the primer binding site and 5′ majorsplice donor site can readily be established by one skilled in the artby reference to the published SIV sequences or for example by aligning avariant SIV to the sequences set out in Table 5.

In some preferred embodiments, a SIV genome has a mutation within thepackaging signal such that the SIV RNA is not packaged within the SIVprotein coating or capsid i.e. the mutation prevents packaging of theSIV genome). Preferably, such an SIV genome is capable of producing anSIV capsid.

The packaging regions of SIV are well known in the art and the skilledperson is readily able to produce packaging deficient mutations usingstandard techniques. In some preferred embodiments, the packagingdefective genome does not contain the SIV packaging sequences whichcorrespond to the segments immediately downstream of the primer-bindingsite and just upstream of the 5′ major splice donor of the SIV genome(residues 849-985 numbered from the 5′LTR) and/or those immediatelydownstream of splice donor and immediately upstream of the gag gene(residues 985-1054 numbered from the 5′LTR).

In some embodiments, the vector may contain nucleotides ranging fromabout 20 bases downstream of the primer-binding site to about 80 basesdownstream of the primer-binding site and still be packaging-deficient(for example, nucleotides ranging from residue 869 to residue 929,numbered from the 5′LTR) and/or about 20 bases downstream of the majorsplice donor to 70 bases downstream (for example, nucleotides rangingfrom residues 1005 to 1054).

Preferably, the packaging sequence absent from the vector comprises allor part of the region between the primer binding site and the 5′ majorsplice site (for example, residues 849-985 numbered from the 5′LTR).

In some embodiments, the packaging sequence absent from the vector inthis region may contain or comprise the 85-base nucleotide sequence ofSEQ ID No. 2 i.e.AGAACTCCTGAGTACGGCCTGAGTGAAGGCAGTAAGGGCGGCAGGAACCAACCACGACGGAGTGCTCCTATAAAGGCGCAGGTCG.

The mutation of the SIV genome may comprise a deletion of:

-   (a) the sequence of SEQ ID NO: 2, or-   (b) a fragment thereof of 5 or more nucleotides in length, or (c) a    variant of either (a) or (b).

A variant of the sequence of SEQ ID NO: 2 may include the correspondingsequence derived from a variant SIV genome, which may be identified forexample by identifying the major 5′ splice donor site, primer bindingsite or gag initiation codon and aligning the sequence of the variant toSEQ ID NO: 2 to identify the corresponding sequence of the variant SIVgenome to SEQ ID No 2. Such a variant sequence may show at least 70%sequence homology to SEQ ID NO: 2, more preferably at least 80%, evenmore preferably at least 90 or 95%.

One preferred variant of SEQ ID NO: 2 may have the sequence of residues854 to 937 of accession number D01065.1.

In preferred embodiments, the packaging signal comprises part or all ofthe region of the genome 5′ to the major splice donor site, for examplea region commencing 90, 80, 70, 60 or 50 nucleotides upstream of (5′ to)the 5′ major splice donor site (at position 985 numbered from the 5′LTR), extending to 40, 30, 20 or 10 nucleotides upstream of (5′ to) the5′ major splice donor site. The mutation may comprise deletion ormutation within this region, for example to modify or delete 5, 10, 15,20, 30, 40, 50 or 60 or more nucleotides from this region, preferablycontiguous nucleotides. For example the packaging deletion may comprisenucleotides 53 to 85 of SEQ ID No 2.

This region of the SIV genome is the structural fold termed DIS and isassociated with a palindromic terminus. In some preferred embodiments,the packaging sequences in this region are mutated to disrupt theformation of the palindromic terminus and thus remove the DIS structure.

In some embodiments, other SIV genomic sequences, such as thosedownstream of the 5′ major splice donor site extending up to the gaginitiation codon may be deleted. Preferably, such sequences are deletedin addition to the mutation of sequences upstream of the major splicedonor. For example, the virus genome may have a deletion or mutation ofall or part of the 50-base segment sequence shown as SEQ ID No. 3, i.e.

GAAATAGCTGTCTTGTTACCAGGAAGGGATAATAAGATAGATTGGGAGAT, or the correspondingsequence from a different SIV strain (e.g. nucleotides 1006-1054 of AccNo: D01065.1).

The number of bases that need to be deleted or mutated can vary greatly.For example, the given 50 or 85-base pair deletions in SIV aresufficient to result in loss of packaging ability.

However, even smaller deletions in this region may result in loss ofpackaging efficiency. A deletion as small as about 5, 10, 15, 20 or 30or more bases in this region and in particular a deletion in the regionof nucleotides 53 to 85 of SEQ ID No 2, or a corresponding sequence, canremove efficient packaging ability. The size of a particular deletion toreduce or abrogate packaging ability can readily be determined based onthe present disclosure by the person of ordinary skill in the art.

A mutation may comprise a deletion or modification of the sequence ofSEQ ID NO: 2. An appropriate modification may comprise a substitution,addition and/or deletion. An appropriate mutation will be one whichleads to a reduction in the ability of viral RNA to be packaged withinan SIV capsid. Preferably, such a mutation will lead to viral RNA notbeing packaged within an SIV capsid.

The mutation may alternatively comprise deletion or modification of afragment of SEQ ID NO: 2 or a variant thereof of 5 or more nucleotidesin length. Such a fragment may be an internal fragment, that is to say,a deletion of 5 or more nucleotides within SEQ ID NO: 2, not includingthe end nucleotides of SEQ ID NO: 2. Such a fragment may be, forexample, 5, 10, 15, 20 or 25 nucleotides in length. In the alternative,the fragment may comprise a fragment of 17 or more nucleotides inlength, selected from any portion of SEQ ID NO: 2 or a variant thereofincluding a terminal fragment thereof. Such a fragment may be, forexample, 15, 25, 35, 45, 55, 65 or 75 nucleotides in length.

Alternatively, larger deletions may be incorporated. Preferably, alarger deletion will comprise the 85 base nucleotide region shown in SEQID NO 2 or a variant thereof and will extend from this location in theSIV genome in one or both directions. Such a deletion may comprise adeletion of, for example, 1, 2, 5, 10, 20, 30, 50 or more bases at oneor both ends of this sequence.

As described above, the vector preferably contains an SIV nucleotidesegment containing a sufficient number of nucleotides corresponding tonucleotides of the SIV genome to express functional SIV gene products,but as described above, should not contain a sufficient number ofnucleotides corresponding to the region between the primer-binding siteand the 5′ major splice donor or between 5′ major splice donor and gaggene to permit efficient packaging of the viral RNA into virions. Inother words, the vector preferably comprises an SIV nucleic acidsequence encoding functional SIV gene products, such as gag and pol, toproduce an SIV capsid as described above, but does not contain an SIVpackaging region which allows efficient packaging of the viral RNA intovirions.

In establishing SIV packaging-defective cell lines, it is preferred thatsuch cell lines do not produce any infectious SIV. Although a cell linetransformed by these packaging-defective deficient vectors would havelow infectivity because the cells are packaging-defective, some RNA canstill be packaged into the virion. Accordingly, it is preferable thatthe SIV nucleotide segment or nucleic acid sequence in the vector doesnot correspond to the entire SIV genome so that, if some of the viralRNA is packaged into the virion, what is packaged will not bereplication-competent virus.

The SIV genome as used herein refers to the viral RNA derived from anSIV. The SIV be derived from any SIV strain, or derivatives thereof.Examples of genomic sequences of different strains of SIV are shown inTable 5. Derivatives preferably have at least 70% sequence homology tothe SIV genome, more preferably at least 80%, even more preferably atleast 90 or 95%. Other derivatives which may be used to obtain theviruses of the present invention include strains that already havemutations in some SIV genes. Other mutations may also be present as setout in more detail below. The position of locations such as the primerbinding site and 5′ major splice donor site can readily be establishedby one skilled in the art by reference to the published SIV sequences orfor example by aligning a variant SIV to the sequences set out anddescribed herein.

Vectors Comprising HIV-2 Packaging Sequences

The vectors comprising HIV-2 packaging sequences may be packaged, asdescribed herein, by the SIV envelope or heterologous viral envelopessuch as the Amphotrophic Murine Leukaemia Virus envelope, VesicularStomatitis Virus G protein (VSV-G) or other Rhabdovirus envelopes. Thesevectors may be capable of being packaged by HIV-1 and/or HIV-2.

The invention encompasses a vector for expression of a heterologous genewhich may be packaged into the SIV genome through the use of HIV-2packaging sequences. Such a vector may comprise any suitable vectorcompatible with the proposed administration or use of the virus, whichhas an HIV-2 packaging sequence incorporated therein. Preferably, thevector is derived from the HIV-2 genome but includes mutation in one ormore HIV-2 genes, for example, to render the HIV-2 genome replicationdeficient.

A suitable HIV-2 vector should contain a sufficient number of HIV-2nucleotides (i.e. contiguous nucleotides from the HIV-2 genome) topermit efficient packaging of the viral RNA into virions.

HIV-2 has been described in a number of references. For example, McCannand Lever (1997) disclose pSVR which is in an infectious proviral cloneof the ROD strain of HIV-2 containing the replication origin of simianvirus 40. HIV-2 nucleotide positions herein are numbered relative to thefirst nucleotide of the viral RNA, that is, the transcript start site isdefined as 1. Other examples of strains of HIV-2 are shown in Table 6.

HIV-2 packaging sequences have also been described in the art (Griffin,S.D.C et al, J. Virol. 2001).

SEQ ID NO: 1 comprises positions 380-408 of the HIV-2 RNA and has beendemonstrated as being important for packaging of HIV-2. The 28 basednucleotide sequence of SEQ ID NO: 1 is: AACAAACCACGACGGAGTGCTCCTAGAA.

Preferably, a HIV-2 vector of the invention comprises an HIV-2 genomewhich comprises at least (a) SEQ ID NO: 1 or a fragment thereof, (b) aninternal fragment thereof of 5 or more contiguous nucleotides in length,or (c) a fragment thereof of 17 or more contiguous nucleotides inlength. SEQ ID NO: 1 also corresponds to residues 378-406 of HIV2 strainROD (M15390.1).

A suitable vector may comprise a complete HIV-2 packaging signal or asequence of SEQ ID NO: 1 comprising one or more modifications. Anappropriate modification may comprise a substitution, addition and/ordeletion. An appropriate modification will be one which retains theability of viral RNA to be packaged within an HIV-2 capsid. The skilledperson can easily determine whether or not this packaging occurs for anygiven sequence.

In some embodiments, the vector may comprise a partially deleted ormodified fragment of SEQ ID NO: 1, or a variant thereof, of 5 or morenucleotides in length. Such a fragment is preferably an internalfragment, that is to say, a fragment of 5 or more contiguous nucleotideswithin SEQ ID NO: 1, not including the end nucleotides of SEQ ID NO: 1.Such a fragment may be, for example, 5, 10, 15, 20 or 25 nucleotides inlength. In the alternative, the fragment may comprise a fragment of 17or more nucleotides in length, selected from any portion of SEQ ID NO: 1or a variant thereof including a terminal fragment thereof. Such afragment may be, for example, 17, 19, 21, 23, 25, or 27 nucleotides inlength.

Alternatively, larger portions of the HIV-2 genome may be incorporated.Preferably, such a larger portion will comprise positions 380-408 of theHIV-2 RNA and will extend from this location in one or both directions.Such a portion may comprise, for example, 1, 2, 5, 10, 20, 30, 50 ormore bases at one or both ends of this sequence. This region of theHIV-2 genome includes a proposed structural fold, and is associated witha palindromic terminus. Preferably the deletion will allow the formationof the palindromic terminus. Preferably the vector will comprise asequence lying between the primer binding site and this proposedstructural fold.

A variant of the sequence identified in SEQ ID NO; 1 is a correspondingsequence derived from a variant HIV-2 genome which may be identified,for example, by identifying the major 5′ splice donor site, primerbinding site or gag initiation codon of a variant HIV-2 genome andaligning the sequence of the variant to SEQ ID NO: 1 or to the sequenceof the HIV-2 genome described in McCann and Lever (supra) to identifythe corresponding sequence of the variant HIV-2 genome to SEQ ID NO: 1.A variant preferably have at least 70% sequence homology to the SEQ IDNO: 1, more preferably at least 80%, even more preferably at least 90 or95%. Sequence homology is discussed elsewhere herein.

The HIV-2 genome as used herein refers to the viral RNA derived fromhuman immunodeficiency virus type 2 (HIV-2). HIV-2 may be derived fromany HIV-2 strain, for example an HIV-2 genome set out in Table 6, orderivatives thereof. Derivatives preferably have at least 70% sequencehomology to the HIV-2 genome, more preferably at least 80%, even morepreferably at least 90 or 95%. Other derivatives which may be used toobtain the viruses of the present invention include strains that alreadyhave mutations in some HIV-2 genes. Other mutations may also be presentas set out in more detail below. The position of locations such as theprimer binding site and 5′ major splice donor site can readily beestablished by one skilled in the art by reference to the publishedHIV-2 sequences or for example by aligning a variant HIV-2 to thesequences set out and described herein.

The packaging sequences which are present in such a vector maycorrespond to those sequences which are mutated to produce a packagingdefective HIV-2 vector. Preferably, a substantial portion of thepackaging signal is included. In a preferred aspect, the packagingsequence comprises the sequence of SEQ ID NO: 1, or a fragment thereofor a variant thereof. All of the HIV-2 sequences described above arepreferred sequences for incorporation into a vector such that the vectorcan be packaged by an SIV capsid or protein envelope.

Alternatively and/or additionally to the packaging sequences describedabove, further HIV-2 packaging sequences may be present in a vector.These sequences may comprise 10, 20, 50, 100, 200, 300 or 400 or morepolynucleotides from a region downstream of the 5′ splice donor site. Ina preferred aspect, these packaging sequences comprise the 5′ part ofgag, preferably comprising the matrix (MA) region of the gag ORF. In apreferred aspect, the packaging sequence comprises the sequence thatlies between positions 553 and 912 of the HIV-2 RNA, or a variantthereof. A variant of such a packaging sequence is a correspondingsequence derived from a variant HIV-2 genome which may be identified,for example, by identifying the major 5′ splice donor site, primerbinding site or gag initiation codon of a variant HIV-2 genome andaligning the sequence of the variant to the sequence of the HIV-2 genomedescribed in McCann and Lever (supra) to identify the correspondingsequence of the variant HIV-2 genome to SEQ ID NO: 1.

These vectors may be useful in efficiently packaging desired geneticsequences and delivering them to target cells. This may be done bypreparing a vector containing a nucleotide segment containing asufficient number of nucleotides corresponding to the packagingnucleotides of HIV-2 (HIV-2 packaging region), a predetermined gene and,flanking the packaging sequence and predetermined gene, sequencescorresponding to a sufficient number of sequences from within and nearthe LTR for packaging, reverse transcription, integration of the vectorinto target cells and gene expression from the vector.

The packaging region preferably corresponds to at least the sequence ofSEQ ID NO: 1. The vector might also comprise the 5′ part of gag,preferably including the matrix (MA) sequence of HIV-2 in order toenhance packaging efficiency. For example, a sufficient number of HIV-2sequences to be packaged, reverse-transcribed, integrated into andexpressed in the target cells would include the U3, R and U5 sequencesof the LTRs, the packaging sequences, the polypurine tract, the primerbinding site and, optionally, the DNA ‘flap sequence. Mutation of thegag initiation codon might be acceptable to avoid translation startingfrom this point whilst still retaining the cis acting gag nucleotidesequence required for packaging. For example, the gag ATG may be changedto ATC by site-directed mutagenesis.

When this vector is used to transfect an SIV packaging-deficient cell,it is the nucleotide sequence from this vector that will be packaged inthe virions produced. These packaged genes may then be targeted tocells.

For example, the vector could contain a sufficient number of nucleotidescorresponding to both 5′ and 3′ LTRs of HIV-2 to be expressed,reverse-transcribed and integrated and a sufficient number ofnucleotides corresponding to the HIV-2 packaging sequences to bepackaged. The vector would also contain a sufficient number ofnucleotides of the gene which is desired to be transferred to produce afunctional gene (e.g. gene segment). This gene can be any gene desired,as described below. The vector may also contain sequences correspondingto a promoter region which regulates the expression of the gene. Thevector may be a self-inactivating vector, for example aself-inactivating retroviral vector. This may comprise a mutation in theU3 region of the 3′LTR of the vector which, after infection of thetarget cell during reverse transcription, is copied so that the 5′ LTRcontains this inactivating mutation, and the long terminal repeatpromoter is inactivated. This leaves any internal promoter to functionindependently of any competition.

Host Cells

In the methods described herein, host cells are generated which produceSIV virus containing a vector for expression of a heterologous gene. Theviruses are produced by co-transfecting a cell, such as a mammaliancell, with a vector which is capable of producing an SIV capsid, forexample a packaging defective SIV vector, and a vector having an HIV-2packaging signal and a heterologous gene.

Preferably, a selected cell line is transformed using at least twodifferent vectors. By co-transfecting a cell with each vector, the cellis able to express all the viral structural and enzymatic proteins andproduce virions.

The, or each, vector may be a self-inactivating vector. As describedabove, this may, for example, comprise a mutation in the U3 region ofthe 3′LTR of the vector which, after infection of the target cell duringreverse transcription, is copied so that the 5′ LTR contains thisinactivating mutation and the long terminal repeat promoter isinactivated. This leaves any internal promoter to function independentlyof any competition.

Selection of particular promoters and polyadenylation sequences canreadily be determined based upon the particular host cell. Preferablythe polyadenylation sequences do not correspond to the 3′LTR.

In some embodiments, one vector may include sequences permittingexpression of proteins upstream of env and the other vector may permitexpression of the remaining proteins. For example, one vector maycontain a nucleotide segment corresponding to a sufficient number ofnucleotides upstream of the gag initiation codon to the env genesequence to express the 5′-most gene products. In other words, thevector may comprise a nucleic acid sequence from the region upstream ofthe gag initiation codon to the env gene sequence which allowsexpression of the 5′-most gene products. The other vector may contain anucleotide segment corresponding to a sufficient number of nucleotidesdownstream of the gag gene sequence and including a functional env genesequence. In other words, the other vector may comprise a nucleic acidsequence from the region downstream of the gag gene sequence whichincludes a functional env gene sequence. Such vectors may be chemicallysynthesised using standard techniques from the reported gene sequence ofthe HIV-2/SIV genome or derived from the many available HIV-2/SIVproviruses using standard recombinant techniques, for example by takingadvantage of the known restriction endonuclease sites in these viruses.

Preferably, each vector comprises a different marker gene. Then, using apre-selected cell line co-transfected with these different vectors, andby looking for a cell containing both markers, a cell that has beenco-transfected with both vectors may be found. Such a cell is able toproduce all of the retroviral proteins. Although virions are produced,the RNA corresponding to the entire viral sequences are not packaged inthese virions.

In some embodiments, more than two vectors may be employed. For example,a gag/pol vector, a protease vector and an env vector may be used.

Retroviruses may be pseudotyped with the envelope glycoproteins of otherviruses. In some embodiments, a vector may contain a sufficient numberof nucleotides to correspond to an env gene from a different retrovirusi.e. the vector may comprise an env gene from a different (non-SIV)retrovirus. Preferably, the 5′LTR of this vector would be of the samegenome as the env gene (i.e. from the same source). Such a vector couldbe used instead of an SIV env packaging-defective vector, to createvirions. By such a change, the resultant vector systems may be used in awider host range or may be restricted to a smaller host range. Forexample, an envelope protein from vesicular stomatitis virus or rabiesvirus may be used to make the vector tropic for many different celltypes.

Any suitable cell line may be used in methods of the invention.Preferably, a mammalian cell line is used, for example CV-1, Hela, Raji,SW480 or CHO.

In order to increase production of the viral cellular products, apromoter other than the 5′ LTR may be used, for example the 5′ LTR maybe replaced with a promoter that will preferentially express genes inCV-1 or HeLa cells. A suitable promoter can easily be determined by theperson of ordinary skill in the art depending on the cell line used.

In order to enhance the level of viral cellular products, enhancersequences may be added to the vector to increase the activity of the LTRand/or promoter. Suitable enhancer sequences can readily be determinedby a person of ordinary skill in the art depending on the host cellline.

By using a series of vectors that together contain a complete retroviralgenome (though a combination of HIV-2 and SIV sequences), cell lines maybe produced that produce a virion that is identical to the SIV virionexcept that the virion does not contain SIV RNA. These virions arereadily obtained from the cells. For example, the cells may be culturedand the supernatant harvested. Depending on the desired use, thesupernatant containing the virions may be used directly or the virionsmay be separated, isolated and/or purified from the supernatant bystandard techniques such as gradient centrifugation, filtering etc.

Attenuated virions as described herein may be extremely useful inpreparing a vaccine. The virions may be used to generate an antibodyresponse to these virions. Pseudotyped virions produced from cell linesco-transfected with retroviral gag/pol and protease genes and containingthe env gene from another virus may be useful in creating a vaccineagainst this other virus.

Methods of Mutation

Mutations may be made in HIV-2 or SIV by homologous recombinationmethods well known to those skilled in the art. For example, HIV-2 orSIV genomic RNA may be transfected together with a vector, preferably aplasmid vector, comprising the mutated sequence flanked by homologousHIV-2 or SIV sequences. The mutated sequence may comprise deletions,insertions or substitutions, all of which may be constructed by routinetechniques. Insertions may include selectable marker genes, for examplelacZ, for screening recombinant viruses by, for example, β-galactosidaseactivity.

The number of bases that need to be deleted or mutated can vary greatly.For example, in SIV, the deletion of the 85-base pair sequence of SEQ IDNO: 2 is sufficient to result in loss of packaging ability. However,even smaller deletions in this region may also result in loss ofpackaging efficiency. A deletion as small as about 5, 10, 15, 20, 30,40, 50, 60, 70 or 80 bases in this region may remove efficient packagingability. The mutation may comprise deletion or modification of afragment of SEQ ID NO: 2 or a variant thereof of 5 or more nucleotidesin length. Such a fragment is preferably an internal fragment, that isto say, a deletion of 5 or more nucleotides within SEQ ID NO: 2, notincluding the end nucleotides of SEQ ID NO: 2. Alternatively, largerdeletions may be incorporated as described above. The size of aparticular deletion can readily be determined by the person of ordinaryskill in the art.

Essential genes may be rendered functionally inactive by severaltechniques well known in the art. For example, they may be renderedfunctionally inactive by deletions, substitutions or insertions,preferably by deletions. Deletions may remove portions of the genes orthe entire gene. For example, deletion of only one nucleotide may bemade, resulting in a frame shift. However, larger deletions aregenerally preferred, for example at least 25%, more preferably at least50% of the total coding and non-coding sequence (or alternatively, inabsolute terms, at least 10 nucleotides, more preferably at least 100nucleotides, most preferably, at least 1000 nucleotides). It isparticularly preferred to remove the entire gene and some of theflanking sequences. Inserted sequences may include the heterologousgenes described below.

Those skilled in the art are well able to construct vectors as describedherein. For further details see, for example, Molecular Cloning: aLaboratory Manual: 3rd edition, Sambrook & Russell, 2001, Cold SpringHarbor Laboratory Press.

Many known techniques and protocols for manipulation of nucleic acid,for example in preparation of nucleic acid constructs, mutagenesis,sequencing, introduction of DNA into cells and gene expression, andanalysis of proteins, are described in detail in Protocols in MolecularBiology, Second Edition, Ausubel et al. eds. John Wiley & Sons, 1992.

Heterologous Genes and Promoters

A vector or virus may be modified to carry a heterologous gene, that isto say a gene or nucleic acid coding sequence other than one present inthe HIV-2 or SIV genome. In particular, vectors are provided which haveHIV-2 derived sequences sufficient to allow packaging of the vector intoa SIV capsid. The vectors may be derived from HIV-2 genomes,incorporating mutations or deletions in one or more HIV-2 genes, or maybe derived from other expression vectors which are modified toincorporate HIV-2 packaging sequences.

The term “heterologous gene” comprises any gene or nucleic acid codingsequence other than one present in the HIV-2 genome. The heterologousgene may be any allelic variant of a wild-type gene, or it may be amutant gene. The term “gene” is intended to cover nucleic acid sequencesencoding a polypeptide and nucleic acid sequences which are capable ofbeing at least transcribed. Thus, sequences encoding mRNA, tRNA and rRNAare included within this definition. The sequences may be in the senseor antisense orientation with respect to the promoter. Antisenseconstructs can be used to inhibit the expression of a gene in a cellaccording to well-known techniques. Sequences encoding mRNA willoptionally include some or all of 5′ and/or 3′ transcribed butuntranslated flanking sequences naturally, or otherwise, associated withthe translated coding sequence. It may optionally further include theassociated transcriptional control sequences normally associated withthe transcribed sequences, for example transcriptional stop signals,polyadenylation sites and downstream enhancer elements.

The heterologous gene may be inserted into for example an HIV-2 vectorby homologous recombination of HIV-2 strains with, for example, plasmidvectors carrying the heterologous gene flanked by HIV-2 sequences. Theheterologous gene may be introduced into a suitable plasmid vectorcomprising HIV-2 sequences using cloning techniques well-known in theart. The heterologous gene may be inserted into an HIV-2 vector at anylocation. It is preferred that the heterologous gene is inserted into anessential HIV-2 gene. Preferably the vector is derived from an HIV-2genome, but includes deletion of one, two or several of the HIV-2 genes,up to the minimal sequences of the HIV-2 genome to provide for packagingand expression of the heterologous gene.

The transcribed sequence of the heterologous gene is preferably operablylinked to a control sequence permitting expression of the heterologousgene in mammalian cells. The term “operably linked” refers to ajuxtaposition wherein the components described are in a relationshippermitting them to function in their intended manner. A control sequence“operably linked” to a coding sequence is ligated in such a way thatexpression of the coding sequence is achieved under conditionscompatible with the control sequence.

A control sequence may comprise a promoter allowing expression of theheterologous gene and a signal for termination of transcription. Thepromoter may be selected from promoters which are functional inmammalian, preferably human, cells. The promoter may be derived frompromoter sequences of eukaryotic genes. For example, it may be apromoter derived from the genome of a cell in which expression of theheterologous gene is to occur. With respect to eukaryotic promoters,they may be promoters that function in a ubiquitous manner (such aspromoters of β-actin, tubulin) or, alternatively, a tissue-specificmanner (such as promoters of the genes for pyruvate kinase). They mayalso be promoters that respond to specific stimuli, for examplepromoters that bind steroid hormone receptors. Viral promoters may alsobe used, for example the Moloney murine leukaemia virus long terminalrepeat (MMLV LTR) promoter or promoters of HIV-2 genes.

The HIV-2 LTR promoter or promoters containing elements of the LTRpromoter, are especially preferred. The expression cassette may furthercomprise a second promoter and a second heterologous gene operablylinked in that order and in the opposite or same orientation to thefirst promoter and first heterologous gene wherein said second promoterand second heterologous gene are the same as or different to the firstpromoter and first heterologous gene. Thus a pair ofpromoter/heterologous gene constructs may allow the expression of pairsof heterologous genes, which may be the same or different, driven by thesame or different promoters. Furthermore, the product of the firstheterologous gene may regulate the expression of the second heterologousgene (or vice-versa) under suitable physiological conditions. Theexpression cassette can be constructed using routine cloning techniquesknown to persons skilled in the art (see, for example, Sambrook et al.,1989, Molecular Cloning—a laboratory manual; Cold Spring Harbor Press).

It may also be advantageous for the promoters to be inducible so thatthe levels of expression of the heterologous gene can be regulatedduring the lifetime of the cell. Inducible means that the levels ofexpression obtained using the promoter can be regulated. For example, inembodiments in which more than one heterologous gene is inserted intothe vector or HIV-2 genome, one promoter may comprise a promoterresponsive to the expression of the second protein and driving theheterologous gene the expression of which is to be regulated. The secondpromoter may comprise a strong promoter (e.g. the CMV IE promoter)driving the expression of the second protein.

In addition, any of these promoters may be modified by the addition offurther regulatory sequences, for example enhancer sequences. Chimericpromoters may also be used comprising sequence elements from two or moredifferent promoters described above, for example an MMLV LTR/HIV-2fusion promoter.

The heterologous gene may encode any desired protein. The heterologousgene may encode, for example, proteins involved in the regulation ofcell division, for example mitogenic growth factors, cytokines (such asα-, β- or γ-interferon, interleukins including IL-1, IL-2, tumournecrosis factor, or insulin-like growth factors I or II), proteinkinases (such as MAP kinase), protein phosphatases and cellularreceptors for any of the above.

The heterologous gene may encode enzymes involved in cellular metabolicpathways, for example enzymes involved in amino acid biosynthesis ordegradation (such as tyrosine hydroxylase), or proteins involved in theregulation of such pathways, for example protein kinases andphosphatases. The heterologous gene may encode transcription factors orproteins involved in their regulation, membrane proteins (such asrhodopsin), structural proteins (such as dystrophin) or heat shockproteins such as hsp27, hsp65, hsp70 and hsp90.

Preferably, the heterologous gene encodes a polypeptide of therapeuticuse, or whose function or lack of function may be important in a diseaseprocess. For example, tyrosine hydroxylase may be useful in thetreatment of Parkinson's disease, rhodopsin may be useful in thetreatment of eye disorders, dystrophin may be useful to treat musculardystrophy, and heat shock proteins may be useful in the treatment ofdisorders of the heart and brain associated with ischaemic stress.Polypeptides of therapeutic use may include cytotoxic polypeptides suchas ricin, or enzymes capable of converting a precursor prodrug into acytotoxic compound for use in, for example, methods of virus-directedenzyme prodrug therapy or gene-directed enzyme prodrug therapy. In thelatter case, it may be desirable to ensure that the enzyme has asuitable signal sequence for directing it to the cell surface,preferably a signal sequence that allows the enzyme to be exposed on theexterior of the cell surface whilst remaining anchored to cell membrane.Suitable signal sequences are well known in the art.

A heterologous gene may encode an antigenic polypeptide useful as avaccine. Preferably such antigenic polypeptides are derived frompathogenic organisms, for example bacteria or viruses, or from tumours.

A heterologous gene may include a marker gene (for example encodingβ-galactosidase, luciferase or green fluorescent protein) or a genewhose product regulates the expression of other genes (for example,transcriptional regulatory factors.

Gene therapy and other therapeutic applications may well require theadministration of multiple genes using the methods described herein. Theexpression of multiple genes may be advantageous for the treatment of avariety of conditions.

Nucleic acid, vectors, viruses and host cells as described herein may beprovided as part of a kit, e.g. in a suitable container such as a vialin which the contents are protected from the external environment. Thekit may include instructions for use, e.g. a method of producing achimeric virus, for example in vitro or in vivo. A kit may include oneor more other reagents required, such as buffer solutions, carriers,etc. Reagents may be provided within containers which protect them fromthe external environment, such as a sealed vial. A kit may includeinstructions for use.

Administration

The vectors, host cells and viruses described herein may be used todeliver therapeutic genes to a human or animal in need of treatment.

A therapeutic gene may for example be inserted into a vector asdescribed above. Subsequently, host cells may be co-transfected in vitrowith a vector comprising the heterologous gene and the HIV-2 packagingsequences and a packaging defective SIV vector. Culturing the cellsleads to production of SIV viral capsids, into which the heterologousgene vectors are packaged through the HIV-2 packaging sequences. Theresultant recombinant virus may, optionally, be purified and/or isolatedbefore use.

In other embodiments, the host cell may be co-transfected in vitro andthen administered to an individual, for example a mammal, in particulara primate such as a human. The host cell may then produce viralparticles comprising heterologous nucleic acid in situ, as describedherein.

In other embodiments, target cells may be co-transfected with the firstand second vectors in vivo. The target cells within the body of theindividual then produce viral particles in situ, as described herein.

While it is possible for the vectors, viruses or host cells to beadministered alone, it is preferable to present it as a pharmaceuticalcomposition (e.g., formulation) comprising at least one active compound,as defined above, together with one or more pharmaceutically acceptablecarriers, adjuvants, excipients, diluents, fillers, buffers,stabilisers, preservatives, lubricants, or other materials well known tothose skilled in the art and optionally other therapeutic orprophylactic agents. Vaccine compositions, in which the heterologousgene encodes an antigenic peptide or protein may be formulated withadjuvants to enhance the immune response generated.

The term “pharmaceutically acceptable” as used herein pertains tocompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgement, suitable for use in contactwith the tissues of a subject (e.g., human) without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. Each carrier,excipient, etc. must also be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation.

Suitable carriers, excipients, etc. can be found in standardpharmaceutical texts, for example, Remington's Pharmaceutical Sciences,18th edition, Mack Publishing Company, Easton, Pa., 1990.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Suchmethods include the step of bringing the active compound intoassociation with a carrier which may constitute one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active compound with liquidcarriers or finely divided solid carriers or both, and then if necessaryshaping the product.

The pharmaceutical composition may be administered to an individual insuch a way that the virus containing the therapeutic gene for genetherapy can be incorporated into cells at an appropriate region of thebody.

The composition may, for example, be formulated for parenteral,intramuscular, intravenous, subcutaneous, intraocular or transdermaladministration.

Formulations suitable for parenteral administration (e.g., by injection,including cutaneous, subcutaneous, intramuscular, intravenous andintradermal), include aqueous and non-aqueous isotonic, pyrogen-free,sterile injection solutions which may contain anti-oxidants, buffers,preservatives, stabilisers, bacteriostats, and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents, and liposomes or other microparticulatesystems which are designed to target the compound to blood components orone or more organs. Examples of suitable isotonic vehicles for use insuch formulations include Sodium Chloride Injection, Ringer's Solution,or Lactated Ringer's Injection. The formulations may be presented inunit-dose or multi-dose sealed containers, for example, ampoules andvials, and may be stored in a freeze-dried (lyophilised) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders,granules, and tablets. Formulations may be in the form of liposomes orother microparticulate systems which are designed to target the activecompound to blood components or one or more organs.

It will be appreciated that appropriate dosages of the vectors, virusesor host cells, and compositions comprising vectors, viruses or hostcells, can vary from patient to patient. Determining the optimal dosagewill generally involve the balancing of the level of therapeutic benefitagainst any risk or deleterious side effects of the treatments of thepresent invention. The selected dosage level will depend on a variety offactors including, but not limited to, the activity of the particularcompound, the route of administration, the time of administration, therate of excretion of the compound, the duration of the treatment, otherdrugs, compounds, and/or materials used in combination, and the age,sex, weight, condition, general health, and prior medical history of thepatient. The amount of compound and route of administration willultimately be at the discretion of the physician, although generally thedosage will be to achieve local concentrations at the site of actionwhich achieve the desired effect without causing substantial harmful ordeleterious side-effects.

Typically, the amount of virus administered is in the range of from 10⁴to 10¹⁰ pfu, preferably from 10⁵ to 10⁸ pfu, more preferably about 10⁶to 10⁷ pfu. When injected, typically 1 to 10 μl of virus in apharmaceutically acceptable suitable carrier or diluent is administered.

Assay Methodologies

Viruses produced as described herein may also be used in methods ofscientific research. Thus, other aspects of the invention relate tomethods of assaying gene function in mammalian cells, either in vitro orin vivo. A method of determining the function of a heterologous gene maycomprise:

-   -   (a) producing virus particles comprising an SIV capsid and a        vector having a heterologous gene packaged via HIV-2 packaging        signals,    -   (b) introducing the resulting virus into a mammalian cell line;        and,    -   (c) determining the effect of expression of said heterologous        gene in said mammalian cell-line.

For example, the cell-line may have a temperature-sensitive defect incell division. When an HIV-2 strain comprising a heterolpgous gene isintroduced into the defective cell-line and the cell-line grown at therestrictive temperature, a skilled person will easily be able todetermine whether the heterologous gene can complement the defect incell division. Similarly, other known techniques can be applied todetermine if expression of the heterologous gene can correct anobservable mutant phenotype in the mammalian cell-line.

This procedure can also be used to carry out systematic mutagenesis of aheterologous gene to ascertain which regions of the protein encoded bythe gene are involved in restoring the mutant phenotype.

Similar methods may be used in animals, for example mice, carryingso-called “gene knock-outs”. A wild-type heterologous gene may beintroduced into the animal using a mutant HIV-2 strain as describedherein and the effect on the animal determined using variousbehavioural, histochemical or biochemical assays known in the art.Alternatively, a mutant heterologous gene may be introduced into eithera wild-type or “gene knock-out” animal to determine ifdisease-associated pathology is induced. In other embodiments, anantisense nucleotide may be introduced using the virus particle of theinvention to create in effect a knock-out animal.

Alternatively, the mutant HIV-2 virus of the invention may be used toobtain expression of a gene under investigation in a target cell withsubsequent incubation with a test substance to monitor the effect of thetest substance on the target gene.

Thus, the methods described herein may be useful for the functionalstudy of genes implicated in disease. Various further aspects andembodiments of the present invention will be apparent to those skilledin the art in view of the present disclosure. All documents mentioned inthis specification are incorporated herein by reference in theirentirety.

The invention encompasses each and every combination and sub-combinationof the features that are described above.

Certain aspects and embodiments of the invention will now be illustratedby way of example and with reference to the figures described above andtables described below.

Table 1 shows a summary of results of virion RNA PCR for GFP and FACSdata on transduced cells with cross-packaged lentiviral vectors

Table 2 shows the cross packaging efficiency of HIV-1 gag-pol (see FIG.1).

Table 3 shows the cross packaging efficiency of SIV gag-pol (see FIG.2).

Table 4 shows the cross packaging efficiency of HIV-2 gag-pol (see FIG.3).

Table 5 shows examples of genomic sequences of SIV strains.

Table 6 shows examples of genomic sequences of HIV-2 strains

EXPERIMENTAL

Overview

The experiments set out below show that HIV-2 helper sequences do notpackage SIV vectors but SIV helper sequences do package HIV-2 vectors.Helper sequences derived from SIV are shown to enable the packaging ofHIV-2 RNA at high levels and permit efficient gene transfer by thepackaged HIV-2 vectors.

Lentiviral Vectors

All SIV constructs are based on the SIV isolate SIVmac32H (GenbankD01065). Numbering refers to positions in the retroviral genome, whereposition 1 is the first base of the 5′ LTR.

The constructs based on HIV-1 and SIV have been previously described(Kaye et al (1998), Strappe et al (2003)). The HIV-1 gene transfervector HR'GFP was modified to include the HIV-1 central polypurine tractor DNA flap sequence. The sequence was PCR amplified and cloned into theunique Clal site upstream of the RRE sequence. The HIV-2 gene transfervector was also modified from the original construct by replacing theSV40-Puromycin construct with a CMV-GFP reporter gene construct (McCannand Lever (1997)). The HIV-2 Gag-Pol construct contains a deletion inthe 5′ untranslated region, which has been shown to abrogate packaging.(Griffin (2001))

Lentiviral Vector Production

Lentiviral vectors were produced by calcium phosphate transfection of293T cells grown in DMEM media and 10% FCS with 7 ug of the genetransfer vector, 7 ug of the Gag-Pol construct, 3 ug of the Revexpresser and 3 ug of the VSV-G heterologous envelope. For HIV-2 and SIVvector production, the Rev expressor was omitted. 24 hours followingtransfection the media was replaced and supernatant containingrecombinant virions was recovered 48 hours post transfection. Virionswere concentrated by ultracenrifugation for 2.5 hours at 25,000 RPM inan SW28 Beckmann rotor. The viral pellet was resuspended in 300 ul oftissue culture media, aliquoted and stored at −70° C.

Lentiviral vectors were titered using a commercially available RT-assay(Cavid Tech, Uppsala) Vector preparations were measured in duplicate andnormalised to a concentration of 8 ng of RT per μl.

Levels of RNA packaging were assessed by RT-PCR of virion associatedRNA. Virion RNA was extracted using the Qiagen Viramp kit from 10 ng ofvirus (RT levels). Following extraction, the RNA was treated with RNasefree DNase for 10 mins at 37° C. and the DNase then inactivated byincubation at 70° C. for a further 10 mins. An aliquot of RNA wasreverse transcribed to cDNA using the Promega Improm RT system with anantisense GFP primer. The cDNA was then serially diluted (1:10) and eachdilution amplified using sense and antisense primers to GFP. Amplifiedproducts were resolved by agarose gel electrophoresis and EtBR staining.

The transduction efficiency of cross-packaged vectors was assessed byFACS analysis of GFP positive cells. A range of viral vectorconcentrations from 40 ng to 4 ng was used to transduce 1×10⁶ SV2C cellsin a six well plate. Viral vector was diluted in DMEM containing 6 ug/mlpolybrene and cells were exposed to virus for 5 hours. The media wasthen replaced and GFP expression was assessed at time periods after 72hours post-transduction ps Glial Cell Culture and Stem Cell Culture

Primary mixed glial cultures were prepared from the brains of newbornrats >3 days old as previously described. Mixed glial cultures werederived from these cells, once they were confluent, by trypsinisation.The cells were then resuspended in DMEM containing 10% FCS and 1% PSFand centrifuged at 10,000 RPM for 5 minutes. The supernatant was removedand cells were resuspended in DMEM/10% FCS and plated onto Poly-D-Lysinecoated coverslips in 24 well plates. Transduction of glial cultures withlentiviral vectors was carried out as described for SV2C cultures. 72hours post transduction, glial cultures were fixed in 4%paraformaldehyde and stored in PBS at 4° C. prior to immunostaining.

Embryonic neuronal stem cell culture was performed as describedpreviously (Wright et al (2003)). Transduction of Stem cell cultures wasperformed with 10 ng of viral vector in stem cell media for 4 hours,followed by replacement of the media. 72 hours post transduction, thecell were fixed in 4% paraformaldehyde, followed by immunostaining forGFP, GFAP and Tubulin.

Immunostaining

Lentiviral vector transduced mixed glial cultures were first blockedusing 3% goat serum in TXTBS (0.2% triton X-100, in Tris BufferedSaline) for one hour. Monoclonal anti GFAP (Sigma, 1:500) and polyclonalgoat anti rabbit GFP (Molecular Probes), 1: 1000) were diluted in TXTBSwith 1% normal goat serum (NGS) for 2 hours. Cells were then washed inTBS for 3×10 minutes. Cells were then incubated with secondaryantibodies, goat anti mouse Alexa (Molecular Probes, 1:500) andbiotinylated goat anti rabbit (Amersham Biosciences, 1:500) for 90minutes. Following a second 3×10 minute wash in TBS, Streptavidin-FITC(Serotec, 1:100) was added in TBS with 1% NGS and Bis-benzamide (Sigma,1:5000). Coverslips were then mounted in Fluorosave reagent(Calbiochem). Cell counts of immunostained mixed glial cultures wereperformed from one edge of the coverslip all the way across to theother, horizontally and vertically. A 0.5 mm2 area was counted every 1.5mm.

Cross-Packaging of Lentiviral RNA

Following concentration of viral vectors by ultracentrifugation, viralvector titre was assessed by the reverse transcriptase assay, whichgives a quantitative measure of RT in ng. The concentration of eachviral vector was normalised to 4 ng/ul following previous optimisation.The levels of RNA packaged in virions were assessed by RT-PCR of thepackaged GFP transgene using specific primers. Virion extracted RNA wasreverse transcribed to cDNA and diluted serially to 1/10, 1/20 and 1/40and then amplified by PCR. Electrophoresis of PCR products reveals alimit of positivity and signal strength. HIV-1 Gag-Pol was found toefficiently package HIV-1 RNA and can also cross package HIV-2 vectorRNA at similar levels, both to a limiting dilution of 1/20. Incomparison, cross packaging of SIV vector RNA by HIV-1 Gag-Pol isreduced and is similar to levels of SIV vector RNA packaged by SIVGag-Pol to a limiting dilution of 1/10.

SIV Gag-Pol was found to efficiently cross package HIV-2 vector RNA to alimiting dilution of 1/40, which is greater than the SIV homologousvector system (1/10) and SIV Gag-pol+HIV-GFP vector system (1/10). Theability of HIV-2 Gag-Pol to cross package HIV-1 and SIV vector RNA issignificantly reduced compared to the homologous HIV-2 system whichshowed similar levels of packaged RNA to the HIV-1 homologous vectorsystem.

Gene Transfer Efficiency of Cross Packaged Vectors

To investigate the gene transfer efficiency of cross-packaged vectors,SVC2 cells were transduced with a range of vector-virion preparations atdiffering concentrations as measured by RT-assay. FIGS. 1 to 3 shows aseries of FACS plots of GFP positive cells following transduction withviral vector and this data is also described in tables 2 to 4.

HIV-1 Gag-Pol was used to package two separate HIV-1 vectors (+/−cPPTsequence), the gene transfer vector containing the cPPT demonstrated anincreased transduction rate of SVC2 cells up to almost a two foldincrease with an input viral vector of 10 ng (FIG. 1; table 2). Transferof 20 ng of an HIV-2 vector packaged by HIV-1 Gag-Pol showed a similartransduction efficiency to that of the HIV-1 cPPT vector packaged byHIV-1 Gag-Pol, suggesting that the HIV-2 cPPT region also contributed toincreased transduction. Transfer of an SIV vector expressing GFP,cross-packaged by HIV-1 Gag-Pol was significantly (almost six fold)lower compared to the homologous HIV-1 viral vector (−cPPT). This mayreflect a low productivity in the SIV vector system, however the genetransfer efficiency of the homologous SIV vector (FIG. 2; table 3) wassimilar to HIV-1 using 4 ng of RT. SIV Gag-Pol demonstrated the abilityto cross package and transfer a HIV-2 GFP vector at levels slightlyhigher than the homologous HIV-1 vector system. This is in contrast tothe lack of gene transfer of a HIV-1 vector packaged by SIV Gag-Pol. Thelevels of HIV-2 vector RNA packaged by SIV Gag-Pol (FIG. 2; table 3) arealso reflected in the high gene transfer efficiency. This packagingrelationship between SIV and HIV-2 would appear to be non-reciprocal,with lower amounts of SIV vector RNA packaged by the HIV-2 Gag-Pol (FIG.3, table 4) and no evidence of any significant gene transfer. Comparingthe HIV-1 and HIV-2 homologous vector systems showed that levels of genetransfer to SVC2 cells were slightly higher for HIV-2 compared to a cPPTnegative HIV-1 vector but lower when compared to the HIV-1 vectorcontaining the cPPT region. HIV-2 Gag-Pol would appear to have noability to cross-package and transfer HIV-1 vector, which is similar toa previous study (Kaye and Lever, 1998) with no significant transductionof SVC2 cells.

Transduction of CNS Cell Types

The cross-packaging and gene transfer relationship between SIV Gag-Poland a HIV-2 vector was verified by transducing rat primary mixed glialcultures. The cultures were transduced with either 40 ng or 20 ng ofviral vector and the efficiency of transduction compared to thatachieved with HIV-1 and HIV-2 homologous vector systems. Cells wereimmunostained for GFP expression and the astrocyte marker GFAP, andcounted.

Transducing the glial cultures with 20 ng of a SIV Gag-Pol+HIV-2 GFPviral vector resulted in GFP positivity in over 30% of cells;approximately 80% of these positive cells were astrocytes. A similartransduction rate was seen with the HIV-1 homologous vector system,which lacks the cPPT sequence, using 20 ng of viral vector. At the sameviral vector concentration, the HIV-2 homologous vector systemtransduced approximately 25% of glial cells with 62% of these cellsstaining for GFAP. The effect of the cPPT sequence on HIV-1 viral vectortransduction is evident with over 60% of glial cell expressing GFP with20 ng of input vector and approximately 58% with long of vector. Insummary, the gene transfer efficiency of the HIV-2 GFP vector crosspackaged by SIV Gag-Pol to glial cells was similar to both the HIV-1 andHIV-2 homologous vector systems (see table 1).

Transduction of human embryonic neuronal stem cells was also performedusing the HIV-1 and HIV-2 homologous vector system and with the SIVGag-Pol /HIV-2 GFP. The transduction efficiency was assessedqualitatively by fluorescence microscopy using 20 ng of viral vector,and the SIV Gag-Pol/HIV-2 GFP cross packaged vector system were found totransduce both astrocytes and neurons post differentiation asdemonstrated by immunostaining with GFAP (astrocytes) and beta-tubulin(Neurons). The cross-packaged vector system performed as well as theHIV-1 and HIV-2 homologous vector systems with astrocytes beingtransduced at a slightly higher efficiency.

In conclusion, a non-reciprocal cross packaging relationship between SIVand HIV-2 has been identified herein. The SIV Gag-Pol/HIV-2 vectorcombination demonstrated equivalent transduction efficiencies in 293Tcells, rat primary astrocytes and embryonic stem cells to that ofhomologous HIV-1 and HIV-2 vector systems.

The methods described herein combine the safety of a vector system inwhich helper and vector sequences are derived from two different viruses(resulting in very low probability of recombination); with the generaladvantages of lentiviral vectors and the specific advantages of HIV-2and SIV sequences. The methods are shown to have a transductionefficiency comparable to the best of other lentiviral systems.

Animal models based on asian macaques and baboons exist for SIV andHIV-2. Thus the SIV/HIV-2 chaemeric vectors described herein may besubjected to direct biosafety testing in animals and subsequently usagein human studies.

REFERENCES

-   Trono D. Gene Therapy 2000, 7, 20-23.-   Connolly JB Gene Therapy 2002, 9, 1730-1734.-   Zufferey R et al. Nat Biotechnol 1997, 871-875.-   Naldini L et al. Science 1996, 272, 263-267.-   Blomer U et al. J Virol 1997, 71, 6641-6649.-   Kordower JH et al. Science 2000, 290, 767-773.-   Dull et al. J Virol, 1998, 72, 8463-8471.-   Lever A et al, J Virol 1989, 63, 4085-4087.-   McCann E.M et al. J Virol, 1997, 71, 4133-4137.-   Strappe PM et al. J Gen Virol, 2003, 84, 2423-2430.-   Kemler I et al. J Virol, 2002, 76, 11889-11903.-   Browning MT et al. J Gen Virol, 2003, 84, 621-627.-   Griffin et al. J Virol 2001, 75, 12058-12069-   Kaye, JF and Lever, AML. J Virol, 1998, 72, 5877-5885.-   White et al. J Virol, 1999, 73, 2832-2840.-   Wright et al J Neurochem. 2003 Jul;86(1):179-950-   Rizvi, TA and Panganibian, A.T (1993) J. Virol, 67, 2681-2688.-   Browning et al (2001) J Virol. 75, 5129-5140.-   Goujon et al (2003) J Virol, 77, 9295-9340.-   Sastry L et al (2003)Mol Ther. 2003;8:830-9.-   Escarpe P et al Mol Ther. 2003,8:332-41.-   Zhao C et al Glia. 2003 , 42,:59-67.-   Baekelandt V et al Gene Ther. 2003,10:1933-40.-   Ruitenberg MJ et al Neurobiol Dis. 2004,15,394-406.-   Ostenfeld T et al J Neurosci Res. 2002, 69, 955-65.-   Certo JL, et al (1998). J. Virol, 72, 5408-5413.-   McCann, E.M and Lever A.M (1997) J virol. 71, 4133-4137.-   Properzi F and Fawcett JW (2004) News physiol Sci, 19, 33-38.-   Tai et al (2004) Curr Opin Pharmacol. 2004 ,4,98-104.-   Englund U et al (2000) Neuroreport. 2000,11, 3973-7.-   Manganini M et al (2002) Hum Gene Ther,13,:1793-807.

Zennou V et al (2001) TABLE 1 Gene Transfer Vector RNA Packaged GFPexpression Gag-Pol (GFP) (Limit of RT-PCR) (Transduced cells) HIV-1HIV-1 10³ +++ HIV-1 HIV-1 (+cPPT) ++++ HIV-1 HIV-2 10³ +++ HIV-1 SIV10² + SIV SIV 10² + SIV HIV-1 10² −(neg) SIV HIV-2 10⁴ ++++ HIV-2 HIV-210³ +++ HIV-2 HIV-1 10² −(neg) HIV-2 SIV 10² −(neg)

TABLE 2 HIV-1 GFP HIV-1 GFP (+cPPT) HIV-2 GFP SIV GFP 40 ng 13770 2136223077 20 ng 6104 12594 11505  8 ng 2122 5639  4 ng 1895 5852 394

TABLE 3 SIV GFP HIV-1 GFP HIV-2 GFP 20 ng  0 0 15792 8 ng 0 0 9232 4 ng2152 14 0

TABLE 4 HIV-2 GFP HIV-1 GFP SIV GFP 20 ng  9621 8 ng 4094 4 ng 1443 4016

TABLE 5 SIV clone/strain Acc No GI number SIVagmSAB-1 U04005.1 gi466229— M58410.1 gi334422 clone 4.41 M31325.1 gi334753 — M32741.1 gi334692isolate STM M83293.1 gi334799 M29975.1 gi1220519 clone 1.5 L03295.1gi334763 SIVMne027 U79412.1 gi2737927 SIVsmE543 U72748.1 gi1695908SIVtan U58991.1 gi1929498 smmPGm AF077017.1 gi3462587 SIVlhoestAF075269.1 gi3342102 US AF103818.1 gi4336706 AF131870.1 gi5106562 SIVcpzAF115393.1 gi6594657 239 M33262.1 gi334647 SIVCPZ AJ271369.1 gi8920373L06042.1 gi294960 Mm251 M19499.1 gi334657 H328 AF316141.1 gi11612154SIVcolCGU1 AF301156.1 gi12657808 1A11 M76764.1 gi334170 M66437.1gi334433 SIVmnd14cg AF328295.1 gi15055096 SIVsmSL92b AF334679.1gi14164886 M27470.1 gi334683 SIVgsn-99CM71 AF468658.1 gi22037883SIVgsn-99CM166 AF468659.1 gi22037893 M30931.1 gi334400 SIVmnd-2AF367411.2 gi26557006 SIVcpzTAN1 AF447763.1 gi27448793 SIVdrl1FAOAY159321.1 gi29367069 SIVmnd5440 AY159322.1 gi29367079 SIVmus-01CM1085AY340700.1 gi37728000 SIVmon-99CMCML1 AY340701.1 gi37728010 SIVdenAJ580407.1 gi39930157 NC_004455.1 gi27311166 NC_001549.1 Gi9627204AF382829.1 Gi21105238

TABLE 6 HIV-2 strain/clone Acc Number GI number 96FR12034 AY530889.1gi47680175 NC_001722.1 gi9628880 MCR35 AY509260.1 gi41056785 MCN13AY509259.1 gi41056775 01JP-IMCJ/KR020.1 AB100245.1 gi32879750 BENM30502.1 gi1332355 7312a L36874.1 gi16905444 J04542.1 gi325654 ALIAF082339.1 gi4007991 EHO U27200.1 gi995584 D00835.1 gi3153166 D205X61240.1 gi60256 U38293.1 gi1845204 M31113.1 gi1339798 ROD M15390.1gi1332361 SBLISY J04498.1 gi1332357 SBL-6699-85 A05350.1 gi345067U22047.1 gi747644 2UC1 L07625.1 gi325762 GH-1 M30895.1 gi325709

1. A process for producing a chimaeric viral vector comprising;culturing a host cell which comprises one or more SimianImmunodeficiency Virus (SIV) nucleic acid sequences capable of producingan SIV capsid and which further comprises a vector comprising a HumanImmunodeficiency Virus type 2 (HIV-2) packaging signal and aheterologous nucleic acid sequence; said vector being packaged in theSIV capsid to produce a chimaeric virus comprising the heterologousnucleic acid sequence.
 2. A process according to claim 1 comprising: (1)infecting the host cell with the vector which comprises the humanImmunodeficiency Virus type 2 (HIV-2) packaging signal and aheterologous nucleic acid sequence, and/or (2) infecting the host cellwith a first vector which comprises the one or more SimianImmunodeficiency Virus (SIV) nucleic acid sequences capable of producingan SIV capsid and a second vector which comprises the humanImmunodeficiency Virus type 2 (HIV-2) packaging signal and aheterologous nucleic acid sequence.
 3. (canceled)
 4. A process forproducing a Simian Immunodeficiency Virus (SIV) encoding a heterologousgene, which process comprises: infecting a host cell with a first vectorwhich is capable of producing SIV capsid and a second vector comprisinga Human Immunodeficiency Virus type 2 (HIV-2) packaging signalsufficient to package the vector in the SIV capsid and a heterologousgene capable of being expressed by the vector; and culturing the hostcell.
 5. A process according to claim 2 wherein the first vector is aSIV vector comprising a mutation within an SIV packaging signal suchthat viral RNA is not packaged within an SIV capsid, and/or the firstvector is a packaging defective SIV vector.
 6. (canceled)
 7. A processaccording to claim 5 wherein said mutation comprises one or more of: adeletion in the region between the primer binding site and the 5′ majorsplice donor site of SIV; a deletion within the DIS structure; adeletion of a sequence of SEQ ID NO: 2 a deletion of a fragment of SEQID NO: 2 5 or more nucleotides in length; a variation of SEQ ID NO: 2 ora fragment thereof of 5 or more nucleotides in length; a deletion in theregion of nucleotides 53 to 85 of SEQ ID NO: 2; a deletion in the regionbetween the 5′ major splice donor and the gag initiation codon; adeletion of SEQ ID NO: 3; a deletion of a fragment of SEQ ID NO: 3 5 ormore nucleotides in length; and/or a variation of SEQ ID NO: 3 or afragment thereof of 5 or more nucleotides in length. 8-12. (canceled)13. A process according to claim 2 wherein the first vector does notcomprise replication-competent SIV.
 14. A process according to claim 2wherein the SIV capsid comprises an envelope protein from a retrovirusother than SIV
 15. A process according to claim 14 wherein the nucleicacid sequence encoding the envelope protein from a retrovirus other thanSIV is operably linked to an 5′ LTR sequence from the same retrovirus16. A process according to claim 2 wherein said second vector comprisesone or more of: (a) a sequence of SEQ ID NO: 1 or a variant thereof; (b)an internal fragment thereof of 5 or more nucleotides in length; (c) afragment thereof of 17 or more nucleotides in length; (d) the matrix(MA) region of the gag ORF or a fragment thereof; (e) nucleic acids 553to 912 of HIV-2 RNA or a fragment thereof; (f) one or more nucleic acidsequences from the 5′ and 3′ LTRs of HIV-2, which direct the expressionand reverse transcription of the second vector and the integration ofthe second vector into the genome of a target cell; and/or (g) apromoter region operably linked to the heterologous gene or nucleic acidsequence. 17-18. (canceled)
 19. A process according to claim 2 whereinthe second vector is replication deficient.
 20. (canceled)
 21. A processaccording to claim 16 wherein the second vector comprises a mutation inthe U3 region of the 3′ LTR of the vector, said mutation being copiedduring reverse transcription such that the long terminal repeat promoteris inactivated
 22. (canceled)
 23. A process according to claim 2 whereinthe said first and/or second vector are; integrated into the genome ofthe host cell, or extra-chromosomal in the host cell.
 24. (canceled) 25.A process according to claim 2 wherein the heterologous gene or nucleicacid sequence encodes a therapeutic protein or peptide, an antigenprotein or peptide.
 26. A process according to claim 2, furthercomprising: isolating and/or purifying the virus comprising theheterologous nucleic acid sequences, and/or formulating the viruscomprising the heterologous nucleic acid sequence with apharmaceutically acceptable excipient.
 27. (canceled)
 28. A processaccording to claim 2 wherein the virus is suitable for infection ofhuman and non-human primate cells.
 29. A process for making a producercell for the generation of chaemeric virus comprising: infecting a hostcell which comprises one or more Simian Immunodeficiency Virus (SIV)nucleic acid sequences capable of producing an SIV capsid, with a vectorcomprising a Human Immunodeficiency Virus type 2 (HIV-2) packagingsignal and a heterologous nucleic acid sequence.
 30. A process accordingto claim 29 wherein the host cell is infected with a first vector whichcomprises the one or more Simian Immunodeficiency Virus (SIV) nucleicacid sequences capable of producing an SIV capsid
 31. A processaccording to claim 29, further comprising one or more of: isolatingand/or purifying the infected cell; and/or culturing said infected cell.32. (canceled)
 33. A virus produced by the process of claim
 1. 34. Avirus according to claim 33 which is capable of infecting human andnon-human primate cells.
 35. (canceled)
 36. A host cell produced by aprocess of claim
 29. 37. A host cell according to claim 36 which is ahuman or non-human primate cell.
 38. A vector system comprising a firstvector which is capable of producing SIV capsid and a second vectorcomprising a Human Immunodeficiency Virus type 2 (HIV-2) packagingsignal sufficient to package the vector in the SIV capsid and a cloningsite suitable for insertion of a heterologous gene capable of beingexpressed by the vector.
 39. A vector system according to claim 38wherein a heterologous gene is inserted into the cloning site.
 40. A kitcomprising a first vector and a second vector of the vector system ofclaim
 38. 41. A method of producing a pharmaceutical composition for usein gene therapy comprising; producing a virus by a process of claim 1,and; formulating the virus with a pharmaceutically acceptable excipient.42. A pharmaceutical composition produced by the method of claim 41.43-48. (canceled)
 49. A method of delivering a therapeutic or antigenicprotein or peptide to an individual comprising; administering to theindividual an effective amount of a virus according to claim 33, or avector system, host cell, or pharmaceutical composition comprising saidvirus.
 50. A method according to claim 49 wherein the individual is ahuman or non-human primate.
 51. A method of transfecting a cell with aheterologous nucleic acid sequence comprising; producing a virus by aprocess according to according claim 1, and; contacting the virus with atarget cell.
 52. (canceled)
 53. A method according to claim 51 whereinthe cell is a CNS cell.
 54. A method according to claim 53 wherein thecell is a glial cell, astrocyte, or neural stem cell.
 55. A method ofdetermining the biosafety of an agent comprising; administering to anon-human primate an effective amount of an agent selected from thegroup consisting of: a virus according to claim 33, a vector systemcomprising said virus, a host cell comprising said virus, or apharmaceutical composition comprising said virus, and determining theeffect of said administration on the primate.
 56. (canceled)